Emphysema consists of a unique pattern of alveolar wall destruction and there is limited therapy against this disease. Cigarette smoking induces oxidative stress and is the most common cause of pulmonary emphysema. Our application seeks to define the mechanism of DJ-1 pathway impairment in this disease. DJ-1 is a multifunctional protein that protects cells from oxidative stress. This depends on posttranslational modifications of cysteine and methionine residues within DJ-1. Kelch-like ECH-associated protein 1 (KEAP1) and CR6-interacting Factor 1 (CRIF1) have been also proposed as factors, which regulate oxidative stress. DJ-1-CRIF1-KEAP1 interaction induces antioxidant defense systems against alveolar epithelial cell injury by cigarette smoke. We propose to study the mechanism of DJ-1 cytoprotective function against human primary alveolar epithelial cell injury by cigarette smoke in vitro and ex vivo. We will determine oxidation of cysteine and methionine residues within DJ-1 using mass spectrometry analysis. We will use mutant constructs to identify, which oxidizable cysteine and methionine residue(s) are critical for DJ-1 function in protecting against injury induced by cigarette smoke. We will focus on cysteine residue at position 106 (Cys-106), which is a sensor of oxidative stress within DJ-1. To further determine the DJ-1 pathway, we will analyze DJ-1, CRIF1 and KEAP1 interacting domains using deletion constructs. We will also use in our studies alveolar type II cells isolated from patients with mild, moderate and severe emphysema. This unique approach will allow us to identify the mechanism of DJ-1 pathway impairment in emphysema progression. We will validate our results in alveolar type II cells in vivo. Wild-type mice will be exposed to cigarette smoke to determine DJ-1 cysteine and methionine modifications by mass spectrometry under oxidative stress conditions. We will also determine whether adenovirus DJ-1 will rescue of alveolar type II cell injury due to cigarette smoke in DJ-1 knockout mice. Study of the mechanism of the DJ-1 pathway impairment in emphysema may lead to the development of novel pharmacological strategies to slow the progression of this disease.